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Biomedical subjects

K E Stein

Publications and source records attributed to K E Stein.

At least 19 recordsLinked to original sources
PubMed1992

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.

Animals↗
PubMed1992

Thymus-independent and thymus-dependent responses to polysaccharide antigens.

Immune responses to polysaccharide antigens are thymus-independent (TI). Conversion of a polysaccharide antigen to a thymus-dependent (TD) antigen by covalent coupling to an immunogenic protein carrier alters the response to the polysaccharide in several important ways. Of primary importance for the prevention of invasive diseases in infants caused by encapsulated bacteria is the shift of the peak antibody response to a much younger age. Another important change is the development of memory B cells primed and ready to respond to either the polysaccharide, as would be encountered during an infection, or to a second dose of the same antigen. Additional immunoglobulin isotypes not seen or seen as a minor component in response to the polysaccharide are also a feature of the TD response. Finally, the diversity of the antibody population is increased after immunization with a TD vaccine compared with that seen after immunization with a TI vaccine.

Animals↗
PubMed1991

VH gene family expression in mice with the xid defect.

Preferential use of particular VH gene families in the response to specific antigens has been demonstrated in several systems. The lack of responses to certain types of antigens, therefore, could be the result of deletion of or failure to express some VH genes. Because CBA/N mice, which carry the X-linked immunodeficiency (xid) gene defect, have been shown to be unresponsive to thymus-independent polysaccharide antigens, it was of interest to examine if this unresponsiveness could be accounted for by abnormal expression of particular VH gene families. Using in situ hybridization on B cell colonies, we determined the expression of nine VH gene families in CBA/CaHN females (genotypically normal), CBA/N males (xid) and females (xid), and (CBA/N x CBA/CaHN)F1 males (xid) and females (phenotypically normal). Our results indicate that VH gene family expression, including the S107 family, in CBA/N males and F1 males, is similar to that of CBA/CaHN and F1 females with predominant expression of J558, the largest gene family, in all individuals. Interestingly, CBA/N female mice, which carry two defective X chromosomes, as a group expressed significantly reduced levels of the J558 gene family, and as individuals showed variation in which family was predominantly expressed. We conclude that the unresponsiveness of mice with the xid defect to polysaccharide antigens can not attributed to a failure to express the nine VH gene families that we examined. Our findings do not support previous studies (Primi, D., and P.-A. Cazenave 1986. J. Exp. Med. 165:357), which found an absence of expression of the S107 family in xid mice.

Animals↗
PubMed1991

A simple method for coating native polysaccharides onto nitrocellulose.

A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.

Animals↗
PubMed1988

Murine immune response to the Neisseria meningitidis group C capsular polysaccharide. I. Ontogeny.

The immune response to polysaccharide Ag develops late in ontogeny and the underlying mechanisms of the infant unresponsiveness are poorly understood. The development of vaccines that will prove efficacious in infants has been hindered by the lack of animal systems suitable for studying immunity to human pathogens. We have examined the BALB/c murine response to the capsular polysaccharide of Neisseria meningitidis group C (MCPS), a homopolymer of alpha(2----9) sialic acid, as a model system for the development of immunity to bacterial polysaccharides in man. We have observed the appearance of natural antibody of both IgM and IgG classes which increases with age, and the transfer of maternal IgG to the offspring. Both the naturally occurring and postimmunization serum responses are restricted to the IgM and IgG3 isotypes, and include antibody titers to both MCPS as well as a natural O-acetyl-negative variant (OAc-). The preimmune anti-OAc- antibodies, in contrast to anti-MCPS, were restricted to the IgM class, whereas after immunization with MCPS both IgM and low titers of IgG3 antibodies to OAc- were produced. These studies demonstrate that the BALB/c mouse strain shows a markedly similar immune profile to that observed in man.

Aging↗
PubMed1988

Murine immune response to the Neisseria meningitidis group C capsular polysaccharide. II. Specificity.

As a means of further understanding the regulation of diversity and the development of protective immunity to the Neisseria meningitidis group C capsular polysaccharide (MCPS), we have generated and characterized, in detail, a panel of mAb against MCPS, a homopolymer of alpha(2----9)-sialic acid. Whereas the serum response to MCPS is restricted to the IgM and IgG3 isotypes, the panel of mAb includes, in addition, both IgG1 and IgG2b. Among 15 mAb of four isotypes, seven different specificities were observed based on direct binding in a fluorescence ELISA and precipitation in gel. Although all the mAb were derived from mice immunized with bacteria encapsulated with the native MCPS (strain C11), only 7 of 15 reacted with MCPS alone. Seven of 15 reacted with a natural O-acetyl-negative variant (OAc-, strain MC19) polysaccharide as well as with MCPS. Five of these reacted as much as 3 logs better with OAc- than MCPS and the other two reacted better with MCPS than OAc-. One mAb appeared to be alpha(2----9)-linkage specific as it reacted not only with MCPS and OAc-, but also with the capsular polysaccharide of Escherichia coli K92, a polymer of sialic acid linked alternately alpha(2----8) and alpha(2----9). None of the mAb reacted with the capsular polysaccharide of E. coli K1, a homopolymer of alpha(2----8)-sialic acid. In general, there was a good correlation between the ability to precipitate Ag in gel and to agglutinate bacteria; however, 3 of 15 mAb, all IgG3, did not conform to this rule in that they precipitated Ag but did not agglutinate bacteria of the relevant capsular specificity. Antibodies of both IgM and IgG isotypes and of both major specificities, MCPS-specific and those binding MCPS and OAc-, were bactericidal for strain C11, whereas only those reactive with OAc- were able to kill strain MC19.

Agglutination Tests↗
PubMed1985

An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides.

A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.

Antibodies, Bacterial↗
PubMed1984

Neonatal administration of idiotype or antiidiotype primes for protection against Escherichia coli K13 infection in mice.

Antibodies directed against the capsular polysaccharides (Ps) of encapsulated pathogenic bacteria can protect the host against infection with such organisms. The immune response to Ps, however, does not develop until relatively late in ontogeny. We have, therefore, studied alternative ways to stimulate anti-Ps antibody responses in neonates, namely priming with idiotype (Id) and anti-Id. We believe that these studies provide the first demonstration of the use of an anti-Id antibody to prime for protection against a bacterial infection and the first demonstration of the ability of a monoclonal anti-Id to prime for protection against any microbial infection. We have used a monoclonal IgM Id, anti-K13 capsular antibody, and a monoclonal IgG1 anti-Id in studies of the effects of administration of anti-Id or Id within 24 h after birth on the ability of mice to respond to subsequent immunization and challenge with live bacteria. These studies show that neonatal administration of 1 micrograms of Id or 50 ng of anti-Id lead to significantly enhanced protection in mice immunized at 4 wk of age and challenged at 5 wk with an intraperitoneal injection of 20-30 LD50 of E. coli 06:K13:H1, as compared with unprimed or antigen (Ps)-primed controls. Mice primed at birth, immunized at 12 wk of age, a time when they can respond fully to Ps itself, and challenged 1 wk later, were still significantly protected by anti-Id priming but no longer showed the effects of Id. We conclude that administration of protective Id early in life may serve a dual function in providing immediate passive protection as well as priming for protective antibodies upon subsequent antigen exposure.

Animals↗
PubMed1984

Anti-immunoglobulin antibodies. V. Age-dependent variation of clones stimulated by polysaccharide TI-2 antigens in 129 and MRL mice spontaneously producing anti-gamma-globulin antibodies.

The nature of the immune response to two conventional polysaccharide thymus-independent (TI) antigens was investigated in two RF-producing mouse strains, the 129/Sv and MRL/1 pr, as well as in their normal congenic counterparts, 129/J and MRL +/+ animals. An age-dependent variation of clones specific for the TI-2 antigens bacterial levan (BL) and alpha 1, 3 dextran B1355 (Dex) was observed in 129/J mice. Surprisingly, the anti-BL and anti-Dex responses observed for young (1-mo-old) 129/Sv mice far exceeded those of their age-matched controls indicating an accelerated ontogenic development of the immune response to TI-2 antigens. A poor response was observed for both MRL +/+ and MRL/1 pr mice after immunization with BL. More importantly, MRL mice, unlike other H-2k, Igh.Ca strains, were unresponsive to Dex in CFA or saline. MRL mice, however, could respond to the T-dependent form of this antigen, Dex-KLH, suggesting that these mice lack the subset of B cells required to respond to TI-2 antigens. Finally, the most striking observation was the occurrence of isotype-specific RF subsequent to immunization with these antigens in animals prone to develop RF, as well as in aged animals that do not spontaneously produce RF.

Aging↗
PubMed1983

Idiotype-antiidiotype regulation. V. The requirement for immunization with antigen or monoclonal antiidiotypic antibodies for the activation of beta 2 leads to 6 and beta 2 leads to 1 polyfructosan-reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies.

The anti-beta 2 leads to 6 fructosan antibodies sharing the idiotypes (Id) of ABPC48 (A48) monoclonal protein represent a silent fraction of the anti-beta 2 leads to 6 fructosan repertoire, since these antibodies cannot be detected during a conventional immune response elicited by bacterial levan (BL). However, the administration at birth of minute amounts of anti-A48 Id antibodies causes a long-lasting activation of A48 Id+-bearing clones. This activation is related to direct interaction of anti-A48 Id antibodies with precursors bearing the A48 Id+ immunoglobulin receptor, since an A48 Id+ response can be transferred with highly purified B cells in lethally irradiated mice. The maturation of these precursors into A48 Id+ anti-beta 2 leads to 6 fructosan antibody-secreting cells requires challenge by the antigen. Isoelectric focusing (IEF) data showed that in 1-mo-old mice an UPC10 (U10)-like spectrotype was observed, whereas in 3-mo-old mice, a new spectrotype binding BL rather than inulin (In) was identified. This spectrotype was observed only in CXBJ mice, the single strain in which an A48 Id+ response was observed. The antigenic challenge can be replaced by a monoclonal anti-A48 Id antibody (i.e., 17-38). Interestingly, in 1-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of A48 Id- anti-beta 2 leads to 6 fructosan-reactive clones with BALB/c type IEF spectrotypes, whereas in 3-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of W3082 IdX+ anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan-reactive clones. In these animals, inhibition of A48 Id+ anti-beta 2 leads to 6 fructosan clones was observed. This antibody probably represents a homobody carrying the internal image of the antigen, which through its paratope suppresses the A48 Id+ response and through its Id activates an A48 Id- anti-beta 2 leads to 6 fructosan response in 1-mo-old mice and in 3-mo-old mice leads to an anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan response dominated by the W3082 IdX.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Immune response to a thymus-dependent form of B512 dextran requires the presence of Lyb-5+ lymphocytes.

Studies of the ontogeny of the immune response to B512 dextran (Dex) show that antibody responses equal to those of adult mice are not attained until 12 wk of age. We have examined the anti-Dex response after immunization with a thymus-dependent antigen isomaltohexaosyl-keyhole limpet hemocyanin (IM6-KLH) and have shown that the development of the cross-reacting anti-Dex response parallels the development of Lyb-5+ B cells. Adult levels of anti-Dex antibody after immunization with IM6-KLH are achieved in mice between 3 and 12 wk of age, a time when Lyb-5+ cells have reached adult levels. Neonatal mice, immunized at 1 d or 1 wk after birth, failed to produce a significant amount of anti-Dex antibodies, although they did produce IM6-specific antibodies after immunization with IM6-KLH. Data, which support the conclusion from these experiments that Lyb-5+ cells are required for an anti-polysaccharide response even when the immunizing antigen is thymus-dependent, include the failure of IM6-KLH to stimulate a normal anti-Dex response in mice with the xid defect and the direct demonstration in normal adult mice that elimination of Lyb-5+ cells from spleens of mice primed with IM6-KLH abolishes the ability of these cells to transfer an anti-Dex response. The data imply that the expressed B cell repertoire in adult animals is skewed such that the vast majority of B cells capable of responding to polysaccharide determinants are in the Lyb-5+ subset.

Animals↗
PubMed1982

The immune response to an isomaltohexosyl-protein conjugate, a thymus-dependent analogue of alpha(1 replaced by 6) dextran.

Two oligosaccharides derived from B512 dextran (Dex), isomaltohexaose (IM6), and isomaltotriose (IM3) have been coupled to keyhole limpet hemocyanin (KLH) and the conjugates studied as thymus-dependent (TD) immunogens analogous in their specificity to Dex. Both IM3-KLH and IM6-KLH stimulate an anti-Dex response in CBA mice. In C57BL mice, only IM6-KLH stimulated an anti-Dex response; IM3-KLH, although immunogenic, failed to elicit the production of anti-Dex antibodies. Similarly, CBA anti-Dex antibodies cross-reacted with both IM3 and IM6 coupled to bovine serum albumin (BSA), whereas C57BL anti-Dex antibodies cross-reacted with IM6-BSA and bound IM3-BSA only very poorly. This reciprocal lack of cross-reactivity between IM3 conjugates and Dex in C57BL mice was controlled by genes linked to the IgH locus, as shown in responses of allotype congenic CBA mice. This genetically regulated fine specificity pattern was overridden when IM6-KLH was used for immunization. C57BL antibodies that bound both Dex and IM3-BSA were produced in response to this antigen. Thus, isomaltohexosyl-protein conjugates provide Dex-like antigens that differ from Dex itself in terms of thymus dependency and the fine specificity of the precursors that are activated. They should provide important tools to study the activation requirements of antigen-specific B cells.

Animals↗
PubMed1980

Anti-immunoglobulin antibodies. I. Expression of cross-reactive idiotypes and Ir gene control of the response to IgG2a of the b allotype.

The anti-allotype antibody response to the b allotypic form of IgG2a is regulated by major histocompatibility complex (MHC)-encoded immune response (Ir) genes. Mice of d, b, p, q, r, and s haplotypes make a strong anti-allotype response on immunization with the CBPC101 myeloma protein (IgG2ab), whereas mice of the k, m, a, a1, u, and z haplotypes made no, or a very poor, response. All responder strains produce anti-IgG2ab antibodies which share common idiotypes (Id) without relation to the allelic forms of the Ig heavy-chain-constant region genes that the responding mice possess. Isoelectric focusing analysis of the anti-allotype antibodies produced in various strains of mice showed that they are of limited heterogeneity and quite similar from strain to strain. Five out of six hybridoma products with specificity for CBPC101 allotype expressed cross-reactive idiotypes (IdX). Two of hybridoma products expressing IdX identify CH3-domain determinants, and one has been assigned a CH2-domain specificity.

Animals↗
PubMed1980

Regulation of the anti-inulin antibody response by a nonallotype-linked gene.

The antibody response to the inulin [(In), beta-(2 leads to 1) fructosan] determinant of bacterial levan [(BL), a beta-(2 leads to 6) polyfructosan that contains beta-(2 leads to 1) branch points] requires the presence of the a haplotype of the Igh gene complex. BALB/c (Igh a) mice immunized with BL produce IgG anti-In antibodies of a single spectrotype by isoelectric focusing analysis. C57BL/6 mice, which possess the b haplotype of the Igh gene complex and which fail to produce anti-In antibodies, nevertheless possess a gene, spectrotype regulation gene 1 (Sr-1), that regulates the isoelectric focusing (IEF) pattern of anti-In antibodies in mice of the a haplotype. Thus, the IEF patterns of anti-In antibodies of (BALB/c x C57BL/6)F1 mice and of B.C8 mice (C57BL/Ka . Igh-Ca) are considerably more complex than those of BALB/c. Backcross analysis indicates that Sr-1 is not linked to the Igh complex, the major histocompatibility complex, or to the genes that code for coat color. Studies of the heterogeneity of anti-In antibodies in recombinant inbred lines and their progeny from matings to BALB/c and C.B20 (BALB/c . Igh-Cb) suggest the existence of other regulatory genes.

Animals↗
PubMed1979

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

Animals↗